Total RNA containing miRNA was isolated from hPASMC and lung tissue samples using QIAzol lysis reagent and column purified using a miRNeasy kit (Qiagen). After quantification with Nanodrop 2000 spectrophotometer (ThermoScientific, Rockford, IL), miRNAs were reversely transcribed using a RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD). For qRT-PCR analysis of miRNA expression, a poly (A) tail was first added to the 3′-end of miRNAs using a Poly (A) Polymerase Tailing Kit (Epicentre Biotechnologies, Madison, WI). Poly (A) tailed-miRNAs were then reversely transcribed using M-MLV Reverse Transcriptase (Invitrogen, Grand Island, NY) with a poly (T) adaptor, which includes a poly (T) sequence and a sequence complementary to the universal primer used in following qRT-PCR analysis. SNORD44, SNORD47 and SNORD48 were used as internal controls. The expression of mRNAs was determined using specific TaqMan primer assays (Applied Biosystems) with GAPDH used as an internal control. Real-time PCR analysis was performed using a CFX384 system (Bio-Rad), and relative changes in mRNA and miRNA expression were calculated after normalization to their respective internal controls using the comparative Ct method.
Poly a polymerase tailing kit
Manufactured by Illumina
Sourced in United States
The Poly(A) Polymerase Tailing Kit is a laboratory equipment product designed for adding a poly(A) tail to the 3' end of RNA molecules. This kit provides the necessary components, including the poly(A) polymerase enzyme, to perform this RNA modification procedure.
Automatically generated - may contain errors
Lab products found in correlation
Megaclear kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
M7g 5 ppp 5 g, by New England Biolabs (3 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Rna clean concentrator 25 kit, by Zymo Research (2 mentions)
Messagemax t7 arca capped message transcription kit, by CellScript (2 mentions)
Rneasy cleanup kit, by Qiagen (2 mentions)
Viia 7 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rnase free water, by Merck Group (2 mentions)
Messagemax t7 arca capped message transcription kit, by Illumina (2 mentions)
Plasmid midi kit, by Qiagen (2 mentions)
Superscript reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
33 protocols using poly a polymerase tailing kit
1
Profiling miRNA and mRNA Expression
2
Cas9 mRNA and sgRNA Synthesis
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Linearized Cas9 plasmids by BamHI enzyme (NEB, USA) were used as templates for Cas9 mRNA synthesis via in-vitro transcription by using mMESSAGE mMACHINE T3 kit (Life technologies, USA). Newly synthesized Cas9 RNA was subsequently 3′-end polyA tail by polyA polymerase tailing kit (Epicentre, USA). sgRNAs were amplified using primer:
F: 5′-TAATACGACTCACTATAGGACCGAGCTCTACTGAGGAACTGTG-3′
R: 5′-AAAAAAGCACCGACTCGGT-3
And then in-vitro transcribed using mMESSAGE mMACHINE T3 kit.
F: 5′-TAATACGACTCACTATAGGACCGAGCTCTACTGAGGAACTGTG-3′
R: 5′-AAAAAAGCACCGACTCGGT-3
And then in-vitro transcribed using mMESSAGE mMACHINE T3 kit.
3
Quantifying mRNA and miRNA Levels in A549 Cells
4
Large-Scale mRNA Preparation and Purification
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RNAs were generated using RiboMax Large Scale Kit (Promega), purified by phenol-chloroform extraction and precipitated by addition of sodium acetate and ethanol as described in standard protocols. Purified RNAs were capped using Vaccinia Capping system (NEB) and then polyadenylated with Poly(A) Polymerase Tailing Kit (Epicentre). Purification from non-incorporated nucleotides was performed with RNeasy Cleanup Kit columns (Qiagen). Concentrations of recovered mRNAs were precisely measured using commercially available fluorescence-based dyes (Invitrogen, Q10210).
5
Analyzing Virus Adaptation Dynamics
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Viruses were passaged on confluent Vero E6 cells infected at a MOI of 0.01 to follow the emergence of compensatory mutations over five sequential rounds of infections. RNA was extracted from infected cells at each passage using TRIzol reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. 3′-rapid amplification of cDNA ends (3′ RACE) was then carried out using a poly(A) polymerase tailing kit (Epicentre Biotechnologies, Tebu-Bio, Le Perray en Yvelines, France). Briefly five micrograms of total RNA was polyadenylated in a 20-μL reaction mixture for 7 min at 37 °C. The RNA was then purified, and reverse transcription was carried out using the oligo(dT) 3′ RACE-adaptor primer (AP) (Invitrogen) or primers specific for the M segment and avian myeloblastosis virus reverse transcriptase (RT) (Promega, Charbonnières, France). This step was followed by polymerase chain reaction (PCR) using the KOD polymerase (Toyobo, Merk, Darmstadt, Germany) and specific primers and/or 3′ RACE-AP. The resulting RT-PCR products were separated by agarose electrophoresis, and the DNA bands with the correct sizes were purified using the QIAquick purification kit (Qiagen, Courtaboeuf, France) and then sequenced according to standard protocols using the BigDye terminator v1.1 kit (Applied Biosystems, Villebon-sur-Yvette, France).
6
Transcriptome Profiling via 5' and 3' RACE
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5′RACE was performed as previously described19 (link), using the 5′RACE System from Invitrogen. cDNA was generated from total RNA using a gene specific primer (GSP1) and then poly(C)-tailed using terminal deoxynucleotidyl transferase. cDNA was then amplified using a nested gene specific primer (GSP2) and the abridged anchor primer from the 5′RACE System.
For 3′RACE, total RNA was first poly(A)-tailed (Poly(A) Polymerase Tailing Kit, Epicentre). cDNA was generated using the 3´RACE System from Invitrogen, with an oligo d(T) adapter primer. cDNA was then amplified using a gene specific primer (GSP3) and the abridged universal amplification primer which anneals to the adapter part of the oligo d(T) primer from the kit. Where possible, PCR products were re-amplified with nested primers (GSP4). Primer sequences are listed in TableS6 .
PCR products were cloned using the pGEM vector system (Promega). Between four and seven plasmid inserts were sequenced per analysis. Alignments of all RACE results are presented in Doc. S1.
For 3′RACE, total RNA was first poly(A)-tailed (Poly(A) Polymerase Tailing Kit, Epicentre). cDNA was generated using the 3´RACE System from Invitrogen, with an oligo d(T) adapter primer. cDNA was then amplified using a gene specific primer (GSP3) and the abridged universal amplification primer which anneals to the adapter part of the oligo d(T) primer from the kit. Where possible, PCR products were re-amplified with nested primers (GSP4). Primer sequences are listed in Table
PCR products were cloned using the pGEM vector system (Promega). Between four and seven plasmid inserts were sequenced per analysis. Alignments of all RACE results are presented in Doc. S1.
7
Rapid Amplification of cDNA Ends (RACE)
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Rapid amplification of cDNA ends (RACE) assays were carried out with the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, #18374-058) and the 3′ RACE System for Rapid Amplification of cDNA Ends (Invitrogen, #18373-019), according to the manufacturer’s protocols. For 5′-RACE, 5 μg of tobacco acid pyrophosphatase (Epicentre Technologies; #RP8092H) treated RNA was reverse transcribed using a sRNA-specific antisense primer and SuperScriptTM Reverse Transcriptase Invitrogen; #18091050). cDNA was then purified, dC-tailed, and used as a template in a PCR reaction with the Abridged Anchor Primer (AAP) and a nested gene-specific primer. 3′-RACE was performed by ligating a poly(A) tail, using a Poly(A) Polymerase Tailing Kit (Epicentre; #PAP5104H) before reverse transcription. Specific cDNAs were then directly amplified by PCR using an Anchor Primer (AP) that targets the poly(A) tail region and a gene-specific primer that anneals to a region of known sRNA sequence. PCR products were cloned into the pGEM®-T Vector System (Promega; #A3600) before sequencing. Primers used in RACE assays are presented in Supplementary Table S2 .
8
Generation of FMNL2-EGFP Fusion Construct
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Template RNA was generated from mouse ovaries with RNA Isolation Kit (Thermo Fisher), then we reversed transcription of these RNA to create cDNA by a PrimeScript 1st strand cDNA synthesis kit (Takara, Japan). Fmnl2-EGFP vector was generated by Wuhan GeneCreate Biological Engineering Co, Ltd. mRNA was synthesized from linearized plasmid using HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G (5′) ppp (5′) G (NEB) and tailed with a poly(A) polymerase tailing kit (Epicentre) and purified with RNA clean & concentrator-25 kit (Zymo Research).
9
In Vitro Transcription of Cas9 Nickase
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Cas9 mutant D10A nickase mRNA and sgRNAs were in vitro transcribed from linear forms of p_1.3_D10A and p_1.1_sgRNA plasmids, respectively. Plasmids were linearised with XbaI and phenol-chloroform purified. mRNA was synthesised using Message Max T7 Arca Capped Message Transcription Kit (Cellscript) and poly-adenylated using poly(A) polymerase Tailing Kit (Epicentre). Single-stranded guide RNAs were synthesised using MEGAshortscript (Ambion).
RNAs were purified using MEGAclear kit (Ambion). RNA quality was assessed using a NanoDrop (Thermo Scientific) and by electrophoresis on 2 % agarose gel containing Ethidium Bromide (Fisher Scientific).
RNAs were purified using MEGAclear kit (Ambion). RNA quality was assessed using a NanoDrop (Thermo Scientific) and by electrophoresis on 2 % agarose gel containing Ethidium Bromide (Fisher Scientific).
10
RT-qPCR Analysis of MALAT1 and miR-140-5p
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Total RNA was extracted from TSCC tissues and cells by TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocols. For the expression analysis of MALAT1, an equal amount of RNA (1 µg) was reverse transcribed into cDNA first strand using M-MLV reverse transcriptase (Promega, Madison, WI, USA), followed by quantitative analysis using FastStart Universal SYBR Green Master (Roche Diagnostics, Mannheim, Germany) with GAPDH as the internal reference. The expression analysis of miR-140-5p was performed by the S-Poly(T) method. Briefly, RNA was first polyadenylated using Poly(A) Polymerase Tailing Kit (Epicenter, Madison, WI, USA), followed by the reverse transcription with M-MLV High-Performance Reverse Transcriptase (Epicenter) via miR-140-5p-RT primer. At last, the expression level of miR-140-5p was detected by miR-140-5p primers (forward and reverse) and Taqman probe with small nucleolar RNA SNORD47 as an endogenous control.
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